Molecular Surveillance of Artemisinin-Based Combination Therapies Resistance in Plasmodium falciparum Parasites from Bioko Island, Equatorial Guinea

ABSTRACT Artemisinin-based combination therapies (ACTs) resistance has emerged and could be diffusing in Africa. As an offshore island on the African continent, the island of Bioko in Equatorial Guinea is considered severely affected and resistant to drug-resistant Plasmodium falciparum malaria. However, the spatial and temporal distribution remain unclear. Molecular monitoring targeting the Pfcrt, Pfk13, Pfpm2, and Pfmdr1 genes was conducted to provide insight into the impact of current antimalarial drug resistance on the island. Furthermore, polymorphic characteristics, haplotype network, and the effect of natural selection of the Pfk13 gene were evaluated. A total of 152 Plasmodium falciparum samples (collected from 2017 to 2019) were analyzed for copy number variation of the Pfpm2 gene and Pfk13, Pfcrt, and Pfmdr1 mutations. Statistical analysis of Pfk13 sequences was performed following different evolutionary models using 96 Bioko sequences and 1322 global sequences. The results showed that the prevalence of Pfk13, Pfcrt, and Pfmdr1 mutations was 73.68%, 78.29%, and 75.66%, respectively. Large proportions of isolates with multiple copies of Pfpm2 were observed (67.86%). In Bioko parasites, the genetic diversity of Pfk13 was low, and purifying selection was suggested by Tajima’s D test (−1.644, P > 0.05) and the dN/dS test (−0.0004438, P > 0.05). The extended haplotype homozygosity analysis revealed that Pfk13_K189T, although most frequent in Africa, has not yet conferred a selective advantage for parasitic survival. The results suggested that the implementation of continuous drug monitoring on Bioko Island is an essential measure. IMPORTANCE Malaria, one of the tropical parasitic diseases with a high transmission rate in Bioko Island, Equatorial Guinea, especially caused by P. falciparum is highly prevalent in this region and is commonly treated locally with ACTs. The declining antimalarial susceptibility of artemisinin-based drugs suggested that resistance to artemisinin and its derivatives is developing in P. falciparum. Copy number variants in Pfpm2 and genetic polymorphisms in Pfk13, Pfcrt, and Pfmdr1 can be used as risk assessment indicators to track the development and spread of drug resistance. This study reported for the first time the molecular surveillance of Pfpm2, Pfcrt, Pfk13, and Pfmdr1 genes in Bioko Island from 2017 to 2019 to assess the possible risk of local drug-resistant P. falciparum.

2. The abstract and importance section need to be overhauled in terms of grammatical, spelling errors and the usage of the wrong verbs and adjectives. Additionally, the grammatical errors make it difficult to understand which message the authors want to convey and can therefore, not be properly evaluated. 3. Line 32: The authors state that prevalence of Pfk13, Pfcrt and Pfmdr1 was 73.68%, 78.29% and 75.66%. Since these are genes that are always present in P. falciparum (prevalence 100%) what are the numbers referring to? Are these numbers based on a specific mutation? 4. Line 96: The manuscript states that the efficacy of DHA/PPQ has not been documented on Bioko Island. Is this also true for the other ACTs? 5. Line 101: The last sentence is a conclusive statement that is not backed up by the data analyzed in the manuscript. To decide on treatment options for malaria patients, additional clinical efficacy studies of the drugs would need to be conducted. Consider revising this sentence to better reflect the outcome of this study. 6. Line 104: The first paragraph could be combined with the following paragraph. 7. Line 112: The sentence states that none of the observed mutations was associated with delayed parasite clearance. Does this refer to previous studies or was this evaluated in this study? For the former, please revise sentence to reflect the reference to former studies and reference them, if the latter, how was this evaluated? Where IC50 data obtained for the isolates? 8. Line 116-117: The sentence talks about the Pfmdr1 and Pfcrt mutations, but the reference to the figure is missing. Please indicate the correct figure reference here. 9. Figure 1: The figure shows the mutations found in pfmdr1, pfcrt and pfk13. The figure does not include the sequence change for pfcrt_K76T. Please describe the schematic gene map and the colors/symbols in the figure legend. C is not mentioned in the text and is not informative for the reader and should be removed from the main figure. Additionally, electropherograms for Pfk13_N194D and Pfk13_S213G are of poor quality and actually showing mixed infection for these two mutations. This should be reflected in the text and analysis. 10. Line 126: The authors state that they used a threshold of 1.5 to define single or multiple copies of Pfpm2. How was this threshold determines? It would be more accurate to have a "positive" control with 2 copies of Pfpm2. I would suggest running a few additional qPCRs with a 2 copy and a 1 copy control to precisely determine the threshold. 11. Line 129-130: The authors speculate that due to increase in Pfpm2 copy number variation drug resistance has emerged in Bioko Island. This is a bold statement that is only made on Pfpm2 copy number variation, experimental susceptibility assays or clinical data is missing. Therefore, I suggest rephrasing this sentence and using more cautious words to describe the potential for drug resistance. Additionally, it would be good to state which drug would be effected. 12. The manuscript uses many different biostatistical analyses for the relatedness and genetic diversity of the isolates. It is important to clarify all the abbreviations used to describe these analyses. Examples are: Line 136: "K value", line 139 "Hd" + "π", Line 141 "dS" and "dN", line 157 "LD", Line 174 "FST index", Line 198 "TCS", Line 204 "FST", Line 182 "EHH" 13. Figure 3: Figure 3 shows the mutation frequency in Pfk13 in African and Asian parasites. Figure 3B: It would be good to describe the frequency scale in the figure legend as there are two different parameters (color and size of square) listed for the mutation frequency. 14. Line 210: The authors state that there is a high rate of malaria transmission on Bioko Island, however, in the introduction it is stated that the transmission rate went down from 45% to 8.5%. Can you elaborate on this? 15. Line 218: The authors state that the study is looking at the efficacy of ACTs by looking at molecular markers. To determine efficacy of the drugs the study would have needed to include either susceptibility data of isolates or clinical efficacy data. I advice to revise this sentence to adequately reflect the topic of the paper e.g. "The data presented here describe the first study on Bioko Island looking at molecular markers associated with ACT drug resistance". 16. Line 233-236: The conclusion for the Pfk13_K189T found in Bioko parasites is that it does not influence artemisinin resistance; but that it might be good to study this mutation further to understand why artemisinin resistance is developing more slowly in Africa. How does this mutation have an influence on artemisinin resistance development? Can you elaborate further on this speculation? 17. Line 249: The authors state that the pfmdr1 ad pfcrt mutation frequency decreased compared to previous findings. However, the authors also noted that this is the first study on Bioko Island. Can you clarify this point? 18. References are overall appropriate for the topic, however, line 253-258 are missing two references. 19. The final conclusion of the paper sates that " Based on our data it is speculated that amodiaquine may be the better artemisinin partner drug than piperaquine and lumefantrine on Bioko Island." I suggest that another sentence will be added calling for additional ex vivo susceptibility and clinical efficacy studies to adequately study this speculation. 20. The authors performed sequencing for Pfk13, Pfcrt and Pfmdr1. The sequencing data should be made available on the NCBI BankIT database.

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The work by Liu et al. investigates the presence of mutations in PfKelch13, the marker associated with resistance to current artemisinin based antimalarials, and known resistance markers for partner drugs like amodiaquine and piperaquine in a region in Bioko Island in Equatorial Guinea from 2017 to 2019. The findings indicate that there is little polymorphism in Pfk13 on Bioko Island with one frequently observed mutation Pfk_K189T that is not associated with artemisinin resistance. Prevalence of Pfcrt and Pfmdr1 mutations associated with resistance to amodiaquine, piperaquine and lumefantrine were fairly high indicating that artemisinin partner drug efficacy might be challenged on Bioko Island. Current recommendations to treat malaria by the WHO are artemisinin combination therapies. Artemisinin resistance is prominent in Asia and first reports suggest association of specific PfKelch13 mutations with delayed parasite clearance in Africa. Surveillance of Pfk13 mutations in African region is of great important. Liu et al. report on surveillance of Pfk13 mutations and partner drugs resistance markers Pfcrt and Pfmdr1 on Bioko Island in Equatorial Guinea. The results suggest little polymorphisms in Pfk13, but presence of resistance markers in Pfmdr1 and Pfcrt. The study adds to the understanding of circling Pfk13 resistance markers in Africa and comparisons with other isolates from Africa and Asia highlight the separate emergence of Pfk13 mutations in Africa and Asia. Comments: 1. The manuscript is well written and conveys the messages clearly and concisely, however, there are several grammatical mistakes, words missing and abbreviations not explained. I suggest looking closely at the manuscript and fixing these issues. A few examples: Line 89, ACTs mentioned twice, line 94 should be Bioko Island reported 2. The abstract and importance section need to be overhauled in terms of grammatical, spelling errors and the usage of the wrong verbs and adjectives. Additionally, the grammatical errors make it difficult to understand which message the authors want to convey and can therefore, not be properly evaluated. 3. Line 32: The authors state that prevalence of Pfk13, Pfcrt and Pfmdr1 was 73.68%, 78.29% and 75.66%. Since these are genes that are always present in P. falciparum (prevalence 100%) what are the numbers referring to? Are these numbers based on a specific mutation? 4. Line 96: The manuscript states that the efficacy of DHA/PPQ has not been documented on Bioko Island. Is this also true for the other ACTs? 5. Line 101: The last sentence is a conclusive statement that is not backed up by the data analyzed in the manuscript. To decide on treatment options for malaria patients, additional clinical efficacy studies of the drugs would need to be conducted. Consider revising this sentence to better reflect the outcome of this study. 6. Line 104: The first paragraph could be combined with the following paragraph. 7. Line 112: The sentence states that none of the observed mutations was associated with delayed parasite clearance. Does this refer to previous studies or was this evaluated in this study? For the former, please revise sentence to reflect the reference to former studies and reference them, if the latter, how was this evaluated? Where IC50 data obtained for the isolates? 8. Line 116-117: The sentence talks about the Pfmdr1 and Pfcrt mutations, but the reference to the figure is missing. Please indicate the correct figure reference here. 9. Figure  Additionally, electropherograms for Pfk13_N194D and Pfk13_S213G are of poor quality and actually showing mixed infection for these two mutations. This should be reflected in the text and analysis. 10. Line 126: The authors state that they used a threshold of 1.5 to define single or multiple copies of Pfpm2. How was this threshold determines? It would be more accurate to have a "positive" control with 2 copies of Pfpm2. I would suggest running a few additional qPCRs with a 2 copy and a 1 copy control to precisely determine the threshold. 11. Line 129-130: The authors speculate that due to increase in Pfpm2 copy number variation drug resistance has emerged in Bioko Island. This is a bold statement that is only made on Pfpm2 copy number variation, experimental susceptibility assays or clinical data is missing. Therefore, I suggest rephrasing this sentence and using more cautious words to describe the potential for drug resistance. Additionally, it would be good to state which drug would be effected. 12. The manuscript uses many different biostatistical analyses for the relatedness and genetic diversity of the isolates. It is important to clarify all the abbreviations used to describe these analyses. Examples are: Line 136: "K value", line 139 "Hd" + "π", Line 141 "dS" and "dN", line 157 "LD", Line 174 "FST index", Line 198 "TCS", Line 204 "FST", Line 182 "EHH" 13. Figure 3: Figure 3 shows the mutation frequency in Pfk13 in African and Asian parasites. Figure 3B: It would be good to describe the frequency scale in the figure legend as there are two different parameters (color and size of square) listed for the mutation frequency. 14. Line 210: The authors state that there is a high rate of malaria transmission on Bioko Island, however, in the introduction it is stated that the transmission rate went down from 45% to 8.5%. Can you elaborate on this? 15. Line 218: The authors state that the study is looking at the efficacy of ACTs by looking at molecular markers. To determine efficacy of the drugs the study would have needed to include either susceptibility data of isolates or clinical efficacy data. I advice to revise this sentence to adequately reflect the topic of the paper e.g. "The data presented here describe the first study on Bioko Island looking at molecular markers associated with ACT drug resistance". 16. Line 233-236: The conclusion for the Pfk13_K189T found in Bioko parasites is that it does not influence artemisinin resistance; but that it might be good to study this mutation further to understand why artemisinin resistance is developing more slowly in Africa. How does this mutation have an influence on artemisinin resistance development? Can you elaborate further on this speculation? 17. Line 249: The authors state that the pfmdr1 ad pfcrt mutation frequency decreased compared to previous findings. However, the authors also noted that this is the first study on Bioko Island. Can you clarify this point? 18. References are overall appropriate for the topic, however, line 253-258 are missing two references. 19. The final conclusion of the paper sates that " Based on our data it is speculated that amodiaquine may be the better artemisinin partner drug than piperaquine and lumefantrine on Bioko Island." I suggest that another sentence will be added calling for additional ex vivo susceptibility and clinical efficacy studies to adequately study this speculation. 20. The authors performed sequencing for Pfk13, Pfcrt and Pfmdr1. The sequencing data should be made available on the NCBI BankIT database.

Replies to Editor and Reviewers,
First of all, we thank both reviewers and editor for your positive and constructive comments and suggestions.
Then, we have read the comments carefully and made correction accordingly. In this letter, we have provided a response to each comment below. In the revised manuscript, all changes were highlighted. Furthermore, the Tables, Figures and supporting information were modified based on the Microbiology Spectrum style.
We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper.
And here we remain the changing trace in revised paper with revisions mode.
Finally, the manuscript was edited for proper English language, grammar, punctuation, spelling, and overall style by Grammarly Premium and AJE Digital Editing.
Once again, we appreciate for Editors/Reviewers' warm work earnestly, and hope that the correction will meet with approval. The detail reply information for the editors and reviewers is as fellows.

With best regards
Yours sincerely

Specific responses to the reviewers's comments
Reviewer #1 ( A: We strongly agree with the reviewer that the resistance could have changed over time. However, the time span and samples number of this study were limited to allow for correlation analysis. In addition, sample collection has been difficult due to the impact of the novel coronavirus epidemic. We will adopt this meaningful suggestion for an in-depth study when the number of subsequent samples reaches a certain number. Here we consider whether resistance and sensitivity analysis can be differentiated by the local rainy and dry seasons. We are grateful to the reviewers for this suggestion, and although we have not explored this time-limited effect in depth, this is an issue that we will focus on in subsequent studies, which will be an interesting topic for future research. 2. Lack of geographical representation. This should be discussed as a limitation. It appears that all samples came from Malabo Regional Hospital and Clinic of the Our previous study analyzed the information underlying the genetic diversity of merozoite surface proteins 1 (MSP-1) and 2 (MSP-2) in P. falciparum from Malabo Regional Hospital, Bioko Island, Equatorial Guinea (PMID: 30526609). The results showed that the MAD20 (9 alleles) family dominated in msp1, followed by the K1 (9 alleles) and R033 (8 alleles) families. In msp2, the FC27 (5 alleles) family was the most frequently detected, followed by the 3D7 (20 alleles) family. It can be seen that although the geographical area of Bioko Island is small, mixed clones of P.
falciparum are still present, which could explain the geographical limitations.
3. Results should be compared against a recent Therapeutic Efficacy Study (TES) that was not referenced. One of the three sites was Malabo. (PMID: 34158055). Do these molecular markers of resistance correlate with clinical efficacy from this recent TES?
A: Thank you for bringing issue to our attention. We agree and have included in the discussion a comparison of this study with the recent efficacy study of Malabo. It is specifically described as follows: In addition, another Pfk13 mutation (Pfk13_R561H) associated with artemisinin resistance has been identified in Africa (PMID: 32747827).
Bioko Pfk13 resistance mutations were not observed in this study, and no significant and Pfmdr1 genes associated with P. falciparum were carried out for P. falciparum isolates.
3. Figure 1 -would be clearer presented as a table. Recommend including the gels (PFE) in a supplement.
A: The sequencing peak map and gel electrophoresis plot make the results more visual and explicit. We have bolded the mutation sites in Figure 1 so that the results can be displayed more prominently and clearly.
4. Figure  A: Here is the prevalence of mutations, which has been modified to "the prevalence of Pfk13, Pfcrt, and Pfmdr1 mutations" (pages 1-2 , lines 32-33 in the revised version).
2. Line 38: "high efficacy" can only be determined with a TES.
A: The reviewer is correct. In the absence of TES, we cannot conclude that "artemisinin and its derivatives remains highly efficacy and amodiaquine considered as the preferential partner drug". We have modified this description to "The results suggested that the implementation of continuous drug monitoring in Bioko island is an essential measure" (page 2, lines 38-39 in the revised version). Once again, we appreciate your warm work earnestly and hope that the correction will meet with approval. All that you mentioned for us will significantly improve the quality of our manuscript. We thank you again for your positive and constructive comments and suggestions.

Line
Reviewer #2 (Comments for the Author): The 6. Line 104: The first paragraph could be combined with the following paragraph.
A: we have merged the above paragraph to the following paragraph as suggested (page 3, lines 104-107 in the revised version).
7. Line 112: The sentence states that none of the observed mutations was associated with delayed parasite clearance. Does this refer to previous studies or was this evaluated in this study? For the former, please revise sentence to reflect the reference to former studies and reference them, if the latter, how was this evaluated? Where IC50 data obtained for the isolates? We rescaled the resolution of the Pfk13_N194D and Pfk13_S213G electropherograms.
Additional descriptions have been added for mixed infections of these two mutations: Using PF3D7_1343700 as the reference sequence, five nonsynonymous mutations (H136N, K189N, K189T, VN193-194V/AD, S213G/A/T) were observed, but none of them was associated with artemisinin delayed parasite clearance (19,20,21). 10. Line 126: The authors state that they used a threshold of 1.5 to define single or multiple copies of Pfpm2. How was this threshold determines? It would be more accurate to have a "positive" control with 2 copies of Pfpm2. I would suggest running a few additional qPCRs with a 2 copy and a 1 copy control to precisely determine the threshold.
A: We used a threshold of 1.5 to define single or multiple copies of Pfpm2 by referring to the reference PMID: 30967148, which suggested that multiple copies vs single copy of Pfpm2, were defined as copy numbers < 1.5 and ≥ 1.5, respectively.
We strongly agree with the reviewer's suggestion of a more accurate "positive" control of 2 Pfpm2 copies and a 2-copy and 1-copy control to precisely determine the threshold. We will further adopt the implementation of this recommendation in the subsequent study.
11. Line 129-130: The authors speculate that due to increase in Pfpm2 copy number variation drug resistance has emerged in Bioko Island. This is a bold statement that is only made on Pfpm2 copy number variation, experimental susceptibility assays or clinical data is missing. Therefore, I suggest rephrasing this sentence and using more cautious words to describe the potential for drug resistance. Additionally, it would be good to state which drug would be effected. 12. The manuscript uses many different biostatistical analyses for the relatedness and genetic diversity of the isolates. It is important to clarify all the abbreviations used to describe these analyses. Examples are: Line 136: "K value", line 139 "Hd" + "π", Line 141 "dS" and "dN", line 157 "LD", Line 174 "FST index", Line 198 "TCS", Line 204 "FST", Line 182 "EHH" A: Thank you for pointing out these issues. We have added specific descriptions of 13. Figure 3: Figure 3 shows the mutation frequency in Pfk13 in African and Asian parasites. Figure  14. A: We have adopted the reviewers' comments and revised the sentence as follows: The data presented in this study are the first molecular investigations conducted for the Pfcrt, Pfk13, Pfpm2 and Pfmdr1 genes on Bioko island and evaluated polymorphic profile of the Pfk13 gene. Accordingly, we aimed to provide some further useful references for assessing ACTs efficacy by monitoring molecular markers related to the drug resistance (page 6, lines 228-232 in the revised version).
16. Line 233-236: The conclusion for the Pfk13_K189T found in Bioko parasites is that it does not influence artemisinin resistance; but that it might be good to study this mutation further to understand why artemisinin resistance is developing more slowly in Africa. How does this mutation have an influence on artemisinin resistance development? Can you elaborate further on this speculation?
A: Pfk13_K189T haplotype analysis revealed a clear evolutionary divergence between P. falciparum in Southeast Asia and Africa. At the same time, the Pfk13 mutant loci in these two regions were significantly different, with the locus in Southeast Asia effectively indicating artemisinin resistance in the local strain, but the resistance locus in the African strain did not effectively indicate the corresponding resistance. It is suggested that artemisinin resistance markers may differ between these two regions and that there are unexplored genes involved in African artemisinin resistance.
The current level of possible artemisinin resistance in Africa is not low, it is just that the available resistance loci are not effectively indicated. Therefore, subsequent new genes and loci for artemisinin resistance need to be re-excavated. This will also be our next key research direction.
17. Line 249: The authors state that the Pfmdr1 ad Pfcrt mutation frequency decreased compared to previous findings. However, the authors also noted that this is the first study on Bioko Island. Can you clarify this point?
A: Although we have done some previous studies on Pfmdr1 and Pfcrt in Bioko Island (PMID: 26325683, PMID: 25348116), our study is the first systematic molecular monitoring of the Pfcrt, Pfk13, Pfpm2 and Pfmdr1 genes in Bioko Island.
Compared with the previous studies, the data in this study are more systematic and comprehensive, which can better provide a reference for the assessment of drug resistance in ATCs.
18. References are overall appropriate for the topic, however, line 253-258 are missing two references.
A: We would like to thank the reviewer for this suggestion. The followed two references were added to the revised manuscript: 19. The final conclusion of the paper sates that " Based on our data it is speculated that amodiaquine may be the better artemisinin partner drug than piperaquine and lumefantrine on Bioko Island." I suggest that another sentence will be added calling for additional ex vivo susceptibility and clinical efficacy studies to adequately study this speculation.
A: We have modified this sentence as suggested: Based on our data, it is recommended that continued monitoring of molecular markers of resistance is necessary in Bioko Island, which might provide baseline prevalence data to guide the use of ACTs. Furthermore, in conjunction with the results of molecular monitoring, additional ex vivo susceptibility and clinical efficacy studies are necessary to further confirm the greater suitability of artemisinin partner drugs such as amodiaquine, piperaquine and lumefantrine (page 7, lines 287-291 in the revised version).
20. The authors performed sequencing for Pfk13, Pfcrt and Pfmdr1. The sequencing data should be made available on the NCBI BankIT database.
A: Based on this suggestion we will subsequently submit and refine the sequencing data of Pfk13, Pfcrt and Pfmdr1 in the NCBI Banklt database.
Once again, we appreciate your warm work earnestly and hope that the correction will meet with approval. All that you mentioned for us will significantly improve the quality of our manuscript. We thank you again for your positive and constructive comments and suggestions.